Chromatin immunoprecipitation analysis of gene expression in the rat uterus in vivo: estrogen-induced recruitment of both estrogen receptor alpha and hypoxia-inducible factor 1 to the vascular endothelial growth factor promoter.

Vascular endothelial growth factor (VEGF) plays a pivotal role in the regulation of microvascular permeability and angiogenesis, processes essential for normal endometrial growth and implantation. Estrogen [17beta-estradiol (E2)], via its receptor (ER alpha), rapidly stimulates VEGF expression in the uterus at the transcriptional level. The VEGF gene promoter, however, lacks a consensus estrogen response element (ERE), and attempts to identify the site through which E2 induces VEGF expression have yielded contradictory results. To address this question, we modified the chromatin immunoprecipitation method to identify transcription factors that interact with the VEGF promoter in the rat uterus in response to E2. Chromatin immunoprecipitation showed that both Sp1 and Sp3 were associated with a proximal, GC-rich region of the promoter before E2 treatment. E2 induced an increase in Sp1 binding and the recruitment of ER alpha, and the coactivator p300 to this region. The association of ER alpha persisted, however, after VEGF mRNA levels had declined again (at 4 h), indicating that other factors might be involved in that expression. Western analysis showed that both the alpha- and beta-subunits of the transcription factor hypoxia-inducible factor 1 (HIF-1), which regulates VEGF expression in response to hypoxia and several hormones and growth factors, were present in the uterus. Furthermore, E2 rapidly induced their recruitment to the -944 to -611 bp region of the VEGF promoter, which contains the hypoxia response element to which HIF-1 binds. This binding was transient, matching the pattern of E2-induced VEGF expression. These results indicate that HIF-1 is an important mediator of E2-induced VEGF expression in the uterus. In addition, E2 also induced a later increase in HIF-1alpha mRNA and protein expression in the uterus, suggesting that it may be required for longer term effects of E2 on the uterus as well. In contrast to the uterus, HEC1A cells cultured in 95% air-5% CO2 (and therefore 20% O2) contained no HIF-1alpha, consistent with the inability of E2 to stimulate the expression of VEGF by these and other cell types in vitro.